The present invention is directed to a method for identifying prion protein which is involved in various transmissible neurological disorders of the central nervous system (CNS) in both humans and animals. Specifically, the method is based on the use of hybridomas or monoclonal antibodies (Mabs) and/or epitope binding fragments thereof prepared against prion protein in order to detect the presence of prion diseases. These antibodies or fragments thereof are suitable for use in highly sensitive immuno-assays for demonstrating the presence of prion protein. Additionally, the invention is also directed to pharmaceutical compositions containing the antibodies or fragments thereof.
A prion is a small infectious protein. It is believed to be the cause of a number of degenerative neurological diseases. These prion caused diseases are collectively hereinafter referred to as xe2x80x9cprion diseasesxe2x80x9d.
Prions were formerly called xe2x80x9cslow virusesxe2x80x9d but are now known to be devoid of nucleic acids and are, therefore, neither viruses nor viroids. The name prion is a contraction of the terms protein and infection. Prions are resistant to inactivation by procedures that modify nucleic acids.
The membrane glycoprotein, now called prion protein (PrP), is involved in the pathogenesis of prion diseases. However, the normal function of PrP and its precise role in disease is not fully understood. It is believed that prion diseases are associated with alterations in PrP. The PrP gene is generally expressed at high levels in neuronal cells of the brain and at lower levels in other tissue such as the heart, lung and spleen. Furthermore, studies indicate that prion diseases are associated with a build up of PrP in and around cells of the brain.
Normal cellular prion protein is encoded by one single gene which can exist in multiple glycoforms with molecular masses existing between 27-40 K. Daltons. The glycoprotein is attached to the cell membrane of mammalian cells by a glycosyl phosphatidyl inositol (GPI) anchor.
Prion caused diseases, or transmissible spongiform encephalopathies (TSE), are neurodegenerative disorders that affect both humans and animals. Prion diseases are referred to as sponiform encephalopathies due to the characteristic of forming holes or pores in cranial tissue.
Development of prion disease may be the result of mutations in the PrP gene. Inherited prion diseases include; Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI) and Gerstmann-Straussler-Scheinker syndrome or disease (GSS) in humans. Prion diseases can also be contracted by an infectious mechanism. This group of diseases includes iatrogenic CJD and a new variant of CJD, which may be the result of transmission of bovine spongiform encephalopathy (BSE, also referred to as xe2x80x9cMad Cowxe2x80x9d disease) from cattle to humans.
The majority of the prion diseases are sporadic disorders. The causes of these sporadic disorders are currently unknown (Parchi, et al., Neurol., 8, 286-293 1995).
The prion diseases present in humans are generally present as a progressive dementia (impairment of intellectual functions) or ataxia (defective muscular coordination). In contrast, scrapie of sheep and bovine spongiform encephalopathy (BSE) are generally manifested as ataxia illness. Sheep and cattle possessing these forms of prion diseases lose their coordination and subsequently have to be destroyed.
The neuropathology of transmissible spongiform encephalopathies (TSE) typically comprises vacuolation of neuronal soma and of neurites and neuronal loss accompanied by reactive astrogliosis. The prognosis of patients having prion diseases is fatal.
Furthermore, the recent discovery of the transmission of bovine spongiform encephalopathy (xe2x80x9cMad Cowxe2x80x9d disease) to humans raises an important health issue concerning the potential spreading of these fatal pathogens from domestic animals to humans. Therefore, there is a strong need for diagnostic agents to detect prion diseases and for therapeutic agents to inhibit infection.
Accumulated evidence suggests that the causative agent underlying prion diseases is a proteinaceous infectious pathogen that lacks nucleic acid (Prusiner, Science. 216, 136-144 1982). Prions differ from conventional bacteria, viruses, and viroids by their unique structure and properties. All prion diseases are believed to share the same basic pathogenic mechanism that involves the conversion of the normal cellular prion protein (PrPc or PrPsen) into a form that is infectious, insoluble in non-ionic detergents and partially resistant to proteases (PrPres or PrPsc). As mentioned above, PrPc is a cell surface protein anchored to the membrane by a GPI anchor.
Along these lines, PrPc and PrPres share an identical amino acid sequence. The conversion of PrPc to PrPres may involve a conformational change of PrPc from a predominantly alpha-helical form to a beta-sheet structure (Pan, et al., Proc. Natl. Acad. Sci. USA, 90, 10962-10966 1993). As a result, the difference between the normal form of PrP and the form associated with diseases may be solely conformational. (Notwithstanding the above, the accumulation within the central nervous system (CNS) of these abnormal PrPres in the brain is a cardinal feature of the prion disease pathology.)
The strongest evidence suggesting that PrPc is essential in the development of prion disease came from studies using PrPc xe2x80x9cknock-outxe2x80x9d mice which are devoid in PrPc and resistant to prion infection (Bueler, et al., Cell. 73, 1339-1347 1993). However, the conditions that trigger and determine the conversion of PrPc to PrPres remain unclear.
Experimental models of inherited prion diseases offer one approach to the study of the PrPc to PrPres conversion. Since many of the pathogenic mutations of the PrP gene (PrPM) have high penetrance, it is likely that the change in PrPM metabolism plays an important role in determining the conversion of PrPM into PrPres. Detailed studies on cell models of inherited prion diseases have underlined the complexity and the diversity of the metabolic changes affecting PrP (Peterson, et al., J. Biol. Chem., 271, 12661-12668 1996).
The concept that TSEs are solely mediated by an infectious proteinaceous agent is not accepted by all investigators. It has been suggested that PrP functions as a cofactor and the development of prion diseases requires another infectious agent (e.g., a virus). Alper, et al., Nature. 214: 764-766 1967. Narang, Proc. Soc. Exp. Biol.Mod. 212: 208-224 1996. The prion hypothesis is also difficult to reconcile with two well established observations: one is the strain specificity of the prion protein and the other is the species restriction of disease transmission. The recent demonstration of the transmission of BSE to primates and mice raises the possibility that at least in some situations the infectious agent is able to surmount the species barrier. Lasmezas, et al., Nature. 381, 743-744 1996. Fraser, et al., Vet. Rec. 123: 472-477 1988. These observations suggest that the transmission of prion disease is a complex process which is still not fully understood.
One of the most puzzling observations in prion infected humans or animals is the lack of a robust inflammatory response during the progression of the disease. Neither humoral nor cell mediated immune responses against the prion protein have been detected in infected humans or animals. Earlier studies suggested that prion infection may result in suppression of the host immune function. Garfin, et al. J. Immunology. 120: 1986-1990 1978. The reasons that prions can evade recognition by the host immune system are not known. Accumulated evidence suggested that host animals may be tolerant to PrPc and PrPres derived from that species. In contrast to many other self proteins, the state of unresponsiveness to the prion protein is not overcome either by infection or by immunization with prion protein in Complete Freund""s Adjuvant (CPA).
A few studies have provided indirect evidence that the host may be able to mount an immunological response against prion. By immunohistochemical staining, T lymphocytes have been observed in the early stages of scrapie (Manuelidis, et al., Science. 277: 94-98 1997). Several inflammatory responses, cytokines and chemokines have been reported to be present in the brain of infected animals (Campbell, et al. J. Virol. 68: 2383-2387 1994. Williams et al., Brain Res. 654: 200-206). Even if immunological responses can be detected in prion infected animals or in patients with prion diseases, the kinetics and magnitude of the responses are different from immune responses observed during infection with conventional microbial pathogens.
Indirect evidence indicates that the host lymphoid system is important in the transmission of prion diseases. In infected animals, the infectious prion protein is found in all components of the lymphoreticular system, including lymph nodes, spleen and Peyer""s patches. (Fraser et al., Nature 226, 462-463 1970. Kimberlin, et al., J. Comp. Path. 89 551-561 1979.) In mice, the infectious prion protein can be detected in the spleen as early as 4 days after intra-peritoneal or surprisingly, intracerebral inoculation. Therefore, propagation of the prion protein in the spleen appears to precede intracerebral propagation.
The important role that leukocytes play in prion disease is further supported by the observation that in vivo activation of the immune system is associated with a shortened incubation period in mice (Dickinson, et al., Nature. 272 54-55, 1978). Furthermore, mitogen activated murine T and B cells are 100 fold more susceptible to in vitro infection with prion protein than non-activated cells (Kuroda, et al. Infect and Immunity. 41, 154-161 1983). Mice with severe combined immunodeficiency (SCID) do not support propagation of the prion protein in the spleen (O. Rourke et al., J. of Gen. Virol. 75: 1511-1514 1994). A recent study suggested that the dendritic cells in the lymphoid tissues may be the source of the infectious prion protein. In another study, it was reported that B cells are critical for disease propagation in mice. Mice lacking B cells are resistant to prion infection by the peripheral route.
In patients with nvCJD, large amounts of PrPres can be detected in the tonsil, an organ rich in antibody producing B cells. (Hill et al, Lancet. 49, 99 1997.) More recently, pathogenic PrP has also been reported to be present in the lymph nodes in the appendix of a patient with nvCJD, eight months prior to the onset of clinical disease. (Hilton, et al., Lancet 352, 703-704 1998.) These observations support earlier findings in animals that pathogenic PrP may accumulate in the lymphoid tissues prior to the development of clinical diseases in the CNS. Whether pathogenic PrP can be detected in other lymphoid tissues in humans has not been studied. Since transmission of both kuru and nvCJD are thought to be transmitted by ingestion of contaminated human tissue or beef, it is likely that the host mucosal immune system is involved in the transmission of these diseases.
While indirect evidence suggests a role for the host immune system in prion infection, the mechanisms by which lymphocytes participate in the establishment and/or propagation of prion diseases are not clear. Some studies suggest that peripheral scrapie infection may occur independently of the lymphoid cells. Alternatively, infection may be spread via the splanchnic complex, prevertebral ganglia or nerve endings in the preitoneal wall. (Aquzzi, Lancet 349: 742-743 1997.) (Kimberlin et al., J. Gen. Virol. 67: 255-263 1989.) These pathways target the infection to the mid-thoracic spinal cord from which it spreads to the CNS and the brain.
Northern blot analysis revealed either the absence, or the presence of very low levels of PrPc mRNA in normal murine spleen. (Caughoy et al., J. Gen Virol. 69: 711-716 1988.) PrPc mRNA is present in murine B lymphocytic cell line and human T lymphocytic cell line. Cashman and colleagues were the first to report the presence of PrPc on normal human peripheral blood lymphocytes that may participate in lymphocyte activation. (Cashman et al., Cell. 61: 185-192 1990.)
However, the mechanism (or mechanisms) by which activated lymphocytes are more effective in promoting prion infection is not known. Progress in understanding prion biology and the pathogenesis of prion diseases has been hampered by the lack of a collection of immunological reagents.
A collection of well-defined monoclonal antibodies is essential for the diagnosis and understanding of many pathological conditions associated with prions. Moreover, the detection of prion diseases is not only dependent upon the availability of highly specific antibodies to PrP, but also antibodies which recognize PrP originating from a number of animal species. The need for a large panel of MAbs is even more critical since Western blotting and immunohistochemistry are the only reliable diagnostic procedures for identifying affected individuals and animals.
Currently, there is only one Mab, i.e. Mab 3F4, that reacts with PrP that has been extensively used to date (Kascsak, et al., J. Virol., 61, 3688-3693, 1987). 3F4 is specific for human and hamster PrPc. However, 3F4 does not recognize PrP from several animal species including mouse, cattle, sheep, monkey and squirrel. This is a significant limitation since transgenic mice are currently the widely used animal models for studying prion disorders. Additionally, Mab 3F4 does not recognize the C-terminal PrPc fragments which are generated during the normal metabolism of PrP or the pathological fragments containing the C-terminal region.
Furthermore, recent studies using PrP transgenic mice and PrPc xe2x80x9cknock outxe2x80x9d mice have dramatically improved applicants understanding of the pathogenesis of prion diseases. A Mab that can react with murine (i.e. mice and rats) PrPc will enhance an investigation ability to elucidate the mechanisms of disease propagation in these animals.
Additionally, 3F4 does not react with cattle or squirrel PrPc. The potential of spreading of prion diseases from infected cattle or squirrel to human warrants the development of Mabs which will allow routine diagnosis and identification of infected animals.
Moreover, some metabolic products of PrPc are important in the pathogenesis of prion diseases. The epitope recognized by Mab 3F4 resides between amino acid 109 and 112. 3F4 does not react with the C-terminal PrP, a normally truncated fragment of PrPc. 3F4 also does not react with all the other pathological fragments located at the C-terminal. Therefore, a panel of anti-PrP Mab recognizing different epitopes, spread allover the PrPc protein will facilitate the ability to detect and monitor the metabolic degradation and recycling of PrPc.
Repeated attempts to generate more Mabs specific for murine PrPc in mice have not been successful. Williamson et. al. reported that immunization of PrP-/- mice with murine PrP in CFA resulted in a strong anti-PrP antibody response. (Williamson et al., P.N.P.S. 93: 7279-7282 1996.) However, these investigators were unable to generate any stable hybridomas. Another group was able to generate Mabs against human prion proteins using PrP-/- mice. (Krasemann et al., Mol. Mod. 2;725-734 1996.) These hybridomas were generated by immunizing the PrP-/- mice with DNA encoding the human prion protein. However, all the Mabs generated were specific for the N-terminal half of the prion protein.
Consequently, it is clear that understanding the biology of PrP and the pathogenesis of prion diseases requires an extensive library of well characterized antibodies to PrP. A collection of diverse Mabs would allow identification of the different metabolic products of PrPc, prpM and PrPres providing new insights into the mechanism of prion conversion. Additionally, these Mabs would also be useful for investigating the involvement of activated lymphocytes in promoting prion infection. Ultimately, these Mabs and/or antibodies thereto will facilitate the diagnosis and/or treatment of prion diseases in humans and in animals.
Applicants have prepared a panel of Mabs specific for PrPc and PrPres. These Mabs cross react with PrPc from other species of mammals including humans, monkeys, cow, sheep, hamster and squirrel. Some of the Mabs selectively recognize PrP metabolic fragments. Additionally, this panel also provides evidence that the PrPc expressed on peripheral blood lymphocytes (PBL) are quantitatively and qualitatively different from PrPc expressed in the brain. Therefore, this panel of Mabs is useful for characterizing and localizating normal, mutant and pathogenic PrP.
More particularly, the present invention is directed to monoclonal antibodies that specifically react with PrP which is involved in the formation of various neurodegenerative diseases. In addition, the invention is directed to the hybridoma cell lines which produce these antibodies. The invention allows for detection of the various forms of PrP using immunological methods comprised of monoclonal antibodies either in solution or in solid phase and specimens taken from a variety of tissue from different mammals.
Along these lines, the present invention is directed to the production of monoclonal antibodies specific for both normal cellular prion protein (PrPc) and pathogenic prion protein (PrPres), and the use of the monoclonal antibodies and associated hybridomas (fused myeloma-lymphocyte cells which produce the monoclonal antibodies) for diagnostic and/or therapeutic use. The monoclonal antibodies can be utilized for a variety of purposes, such as identifying, enumerating, localizing, and isolating PrP though a number of diagnostic means.
The invention also relates to the use of the specific monoclonal antibodies to examine the presence or absence of PrP and PrPres from tissues, e.g. brain, digestive organs, leukocytes, etc., from body fluids in mammals, and from urine and feces by immunoassay. The monoclonal antibodies of the present invention recognize PrP from multiple species including human, mouse, cattle, sheep and squirrel. Therefore, for the first time, it is possible to study and compare the expression of prion proteins from different animals.
Furthermore, since the monoclonal antibodies of the invention also react with sheep, bovine and squirrel PrP, these monoclonal antibodies are useful for diagnosing scrapie and BSE in infected cattle and squirrels.
In another aspect, the monoclonal antibodies of the present invention can distinguish different prion glycoisoforms in western blotting. This is particularly important since the degree, and the nature, of the PrPc glycoforms can modulate the pathogenesis of prion disease.
In an additional aspect, the monoclonal antibodies (and/or antibodies thereto) of the invention can be utilized with a variety of immunoassay and labeling techniques known to those skilled in the art, i.e. competitive and non-competitive assays, sandwich assays, etc. for diagnostic purposes.
In a further aspect of the invention, a diagnostic kit is provided for detecting prion proteins or antibodies thereto. The kit employs at least monoclonal antibodies of the invention and/or antibodies thereto.
In still another aspect, the invention provides monoclonal antibodies and/or antibodies thereto for treatment of prion diseases and/or for the development of therapeutic agents thereto.